A. Incidence and Course of HSV Infection
Herpes Simplex Virus (HSV), also known as herpesvirus hominis, is classified into two (2) types, 1 and 2. HSV-1 is transmitted by physical contact, such as kissing, and is thus spread among family members and friends. About half of all babies in the United States are born with IgG antibodies to this agent which is transmitted across the placenta. As this immunity dissipates, new infections are acquired until, by age 45, close to 70% of people have become serologically positive--most without ever experiencing signs of disease, others after one or several episodes of fever blisters or cold sores.
In contrast, HSV-2, also called genital herpes, is transmitted during birth or by sexual contact. The latter's incidence rises with the number of sexual partners and has therefore greatly increased in today's society. Compared with HSV-1, genital herpes is less prevalent overall but is likewise cumulative with age. In addition, genital herpes has engendered considerable anxiety because there is tenuous evidence that it may contribute to the causation of cervical cancer, and because of the risk of vertical transmission during childbirth inducing serious disease. Infections with both causative agents are difficult to prevent; and there is as yet no proven vaccine for the prophylaxis of genital herpes. Moreover, an ocular vaccine for the prophylaxis of ocular HSV has not been tried in humans.
Turning specifically to HSV-1, it is the most common infectious cause of blindness in industrial nations. Nesburn, A. B., Report of the corneal disease panel: Vision Research: A national plan 1983-1987. Vol. II, part III, edited by Nesburn, A. B., St. Louis, Mo. The often prolonged ocular disease results in considerable visual morbidity, medical expense and loss of productivity in otherwise healthy individuals. Approximately 500,000 cases of ocular HSV-1 are diagnosed annually in the United States alone; and 25% to 45% of these cases may be expected to recur within 1 to 2 years after the primary disease episode. Nesburn, A. B., Report of the corneal disease panel: Vision Research: A national plan 1983-1987. Vol. II, part III, edited by Nesburn, A. B., St. Louis, Mo. Of note, the majority of cases diagnosed as primary HSV are actually recurrent infections, as the patient may not recall the antecedent attack. Recurrence is therefore the hallmark of HSV infection.
Following primary infection with herpes simplex virus, the virus establishes a life long latent infection in sensory nerves. At various times during the life of the latently infected individual, the virus may reactivate, travel back to the original peripheral site of infection, and produce recurrent disease. It is these repeated recurrent infections that are responsible for the vast majority of clinically important herpes simplex infections. Therefore, in addition to reducing the incidence and severity of primary infection, an ideal herpes vaccine should also prevent the establishment of latency, thereby eliminating recurrent disease.
After primary HSV infection occurs, the virus can travel in the nerves to the neurons in the trigeminal ganglia, where it then persists throughout life. This critical factor presently makes the herpes simplex infection an incurable disease, since the virus eventually may travel back down these nerves and reinfect the part of the body innervated by that nerve. Various trigger mechanisms such as trauma, fever, sunlight exposure or stress may initiate the reactivation process. This latency-reactivation-recurrence cycle results in ocular virus shedding despite a good local ocular IgA response to the virus. Klein, R. J., Reinfections and site-specific immunity in herpes simplex virus infections. Vaccine, 7:380-381 (1989). Once HSV has recurred in the eye, corneal disease and stromal scarring can follow, resulting in corneal blindness. Over 1,000 corneal transplants per year are currently performed in the U.S. as a direct result of HSV scarring. Hence, on recovering from the initial HSV infection, the stage is set for reinfection from one's own herpes virus for the remainder of the individual's life.
Since recurrences continue throughout the lifetime of the infected individual, it is clear that natural HSV infection affords insufficient protection against HSV recurrences. Moreover, individuals infected with one HSV serotype are only partially protected against subsequent infection with the other serotype; while individuals with non-ocular HSV-1 are not protected against subsequent ocular HSV-1 infection. Virus from a recurrent lesion on the body can be transferred to the eye, which is thought by some to be a common mode of contracting ocular infections. Because repeated recurrences of HSV do not elicit an immune response that prevents additional recurrences, there is a critical need to elicit a stronger, or perhaps a different immune response than that elicited by natural HSV-1 infection.
With further regard to immune protection, it appears that both antibody and cell-mediated immunity (CMI) are important in the control of HSV infection (Stanberry, L. R. et al., Herpes simplex virus glycoprotein treatment of recurrent genital herpes. J. Infec. Dis., 157:156-63 (1988); Kern, A. B. et al., Vaccine Therapy in Recurrent Herpes Simplex, Arch. Derm., 89:844-845 (1964); and Frenkel, L. et al., A randomized double blind, placebo-controlled phase 1 trial of a herpes simplex virus purified glycoprotein (gD1) vaccine. Interscience Conf. on Antimicrobial Agents & Chemo., 206 (1990), incorporated herein by reference), although CMI may play a larger role. Patients with defects in CMI generally have more severe infection than those with impaired humoral immunity (Berman, P. W. et al., Efficacy of Recombinant Glycoprotein D Subunit Vaccines on the Development of Primary, Recurrent, and Latent Genital Infections With Herpes Simplex Virus Type 2 in Guinea Pigs. J. Infec. Dis., 157(5):897-902 (May 1988); Blacklaws, B. et al., Immunogenicity of herpes simplex type 1 glycoproteins expressed in vaccinia virus recombinants. Virology, 177:727-736 (1990); Spear, P. G., Glycoproteins specified by herpes simplex virus. In: The herpesviruses, edited by Roizman, B., New York, Plenum Press, pp. 315-356 (1985); Narrild, B., Humoral response to herpes simplex virus infections. In: The herpesviruses, edited by Roizman, B., New York, Plenum Press, pp. 69-86 (1985); and Sarminto, M. et al., Membrane proteins specified by herpes simplex virus III. Role of glycoprotein VP7 (B2) in virion infectivity. J. Virol., 29:1149-58 (1979), incorporated herein by reference); whereas patients with frequently recurring HSV have high titers of anti-HSV antibodies. Ophthalmologists have also demonstrated that patients with exuberant immune responses, such as atopes, develop the worst clinical manifestations of stromal herpetic keratitis. Whereas immunosuppressed patients, in contrast, show exacerbated epithelial keratitis but minimal stromal disease. Hence, immunotherapy capable of inducing a specific higher than normal cellular immune response is needed to combat recurrent ocular HSV infections.
Another factor attributing to recurrent ocular HSV infection is the absence of blood vessels in the cornea. Because the cornea is devoid of blood vessels, systemic immune responses have thus far been inefficient at providing protection from antigenic insults there. Stanberry, L. R. et al., Heterologous Versus Homologous Herpes Simplex Virus Glycoprotein Immunotherapy of Recurrent Genital Herpes. Pediatr Res., 25:191A, Part 2 (1989). Consequently, local immunity may be particularly important in protection against ocular HSV. There is therefore a need to develop improved systemic vaccines for ocular HSV, and to develop local ocular immunotherapy to augment the immune response and control recurrent ocular HSV infection.
Currently, commercial HSV vaccine development is directed exclusively to the problem of genital HSV-2. There is minimal effort directed to combat ocular HSV-1. Yet the development of a therapeutic vaccine, that is, a vaccine to reduce HSV ocular recurrences, would greatly alleviate what is now the most frequent serious viral eye infection in the U.S. and a major cause of viral induced blindness in the world. Likewise, the development of a prophylactic vaccine to protect against infection by HSV would similarly be of tremendous importance to reducing the incidence of viral induced blindness. The present invention satisfies these needs and provides related advantages as well. The disclosures of all publications cited herein are expressly incorporated by reference.
B. DNA Technology
Recombinant DNA and associated technologies can be applied to effectively provide the large quantities of high quality bioactive HSV glycoproteins and proteins required for a therapeutic or prophylactic HSV vaccine.
DNA technology involves in part, producing a replicable expression vehicle by the DNA recombination of an origin of replication, one or more phenotypic selection characteristics, an expression promoter, a heterologous gene insert and remainder vector. The resulting expression vehicle is introduced into cells by transformation and large quantities of the recombinant vehicle obtained by growing the transformant. Where the gene is properly inserted with reference to portions which govern the transcription and translation of the encoded DNA message, the expression vehicle may produce the polypeptide sequence for which the inserted gene codes. This process of producing the polypeptide is called "expression." The resulting product may be obtained by lysing the host cell, and recovering the product by appropriate purification.
A wide range of host cells can be used, including prokaryotic and eukaryotic organisms. In addition to microorganisms, cultures of cells derived from multicellular organisms, whether vertebrate or invertebrate, may also be used as hosts.
C. Definitions
As used in this disclosure, the following terms are to be understood in relation to the following definitions.
ADJUVANT: a substance that enhances, nonspecifically, the immune response to an antigen. An adjuvant is usually administered with antigen, but may also be given before or after antigen. Adjuvants disclosed within the subject invention include but are not limited to, alum, Freund's, MTP-PE, ISCOMs, Quil A and liposomes. PA1 ALUM: antigen absorbed into floccules of aluminum salts. Alum is the only adjuvant currently approved by the FDA for human use. PA1 EXPRESSION VECTOR: a vehicle used to carry inserted foreign (heterologous) DNA for the purpose of producing more material or a glycoprotein or protein product. "Expression vector" includes vectors which are capable of expressing the DNA sequences it contains, where such sequences are operably linked to other sequences capable of effecting their expression. Any DNA sequence which is capable of effecting expression of a specified DNA code disposed within the sequence is included in this term as it is applied to the specified sequence. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids; however, this invention is intended to include other forms of expression vectors which serve equivalent functions and which subsequently become known in the art. PA1 FREUND'S: A water-in-oil emulsion. There are two forms of Freund's adjuvant, depending on the presence or absence of killed Mycobacteria. Complete Freund's adjuvant contains Mycobacterium tuberculosis, or other Mycobacteria strains. Weak antigens may be rendered more immunogenic when incorporated in complete Freund's adjuvant. Incomplete Freund's adjuvant lacks Mycobacteria and is less stimulatory. PA1 GLYCOPROTEIN: a class of compounds in which protein is combined with carbohydrate. PA1 MTP-PE: muramyl tripeptide phosphatidyl ethanolamine, a new proprietary adjuvant developed by CIBA and refined by Chiron. It is a potent and well tolerated derivative of Freund's, and has proven to be much more effective than alum. PA1 ISCOM (Quil A): immunostimulating complexes comprising purified proteins and the glycoside Quil A to form a honeycomb like structure that exhibits strong adjuvant activity. PA1 IMMUNOTHERAPY: enhancement of an immune response by any one or more of a variety of adjuvants incorporating one or more antigens. PA1 LIPOSOMES: synthetic lipid vesicles consisting of phospholipid bilayers surrounding one or more aqueous compartments. Antigens can be imbedded in the liposomes for induction of immune responses. PA1 PLASMID: circular double stranded DNA which, in vector form, is not bound to the chromosome. PA1 PROMOTER: a region of DNA involved in the binding of RNA polymerase to initiate transcription. PA1 VACCINE: a composition which produces active immunity. A vaccine is comprised of materials from microorganisms that contain antigens in an innocuous form with or without one or more adjuvants. The materials may comprise antigenic determinants and/or subunits from the microorganism and may be in the form of glycoproteins or proteins. PA1 PROPHYLACTIC VACCINE: an active immunity inducing composition given to naive individuals to prevent or ameliorate primary infection and prevent the establishment of latent infection. PA1 THERAPEUTIC VACCINE: an active immunity inducing composition given to individuals with latent or recurrent infection to reduce or minimize recurrences. PA1 SYSTEMIC VACCINE: a composition for inducing active immunity relating to the entire individual as distinguished from any one individual area. PA1 LOCAL VACCINE: a composition for inducing active immunity relating to one individual area as distinguished from the entire individual.